Process for producing purified anthocyanin and crystalline anthocyanin

ABSTRACT

Provided are a process for producing purified anthocyanidin glucoside in which a rhamnose end of anthocyanidin rutinoside is cleaved using rhamnosidase to convert the anthocyanidin rutinoside component into anthocyanidin glucoside, the anthocyanidin glucoside component being then purified and isolated; or a crystalline anthocyanidin glucoside salt obtained by further crystallizing the purified anthocyanidin glucoside and a process for producing the same.  
     Also provided are a process for producing purified anthocyanidin rutinoside in which a glucose end of anthocyanidin glucoside is cleaved using β-glucosidase to degrade and remove the end, the anthocyanidin rutinoside component being then purified and isolated; or a crystalline anthocyanidin rutinoside salt obtained by further crystallizing the purified anthocyanidin rutinoside and a process for producing the same.

TECHNICAL FIELD

[0001] The present invention relates to a process for producing purifiedanthocyanin from anthocyanin derived from natural product and a processfor producing crystalline anthocyanin by further crystallizing purifiedanthocyanin, and a crystalline anthocyanin prepared by theaforementioned process.

[0002] More particularly, the present invention relates to a processwhich facilitates the subsequent purification and crystallization stepsby enzymatically converting or removing anthocyanidin rutinoside oranthocyanidin glucoside constituting anthocyanins to decreaseanthocyanidin rutinoside or anthocyanidin glucoside.

BACKGROUND ART

[0003] Anthocyan is a generic term for anthocyanidin, which has abackbone represented by the following formula (I), in combination withanthocyanin, which is a glycoside formed by binding of saccharide toanthocyanidin. (I)

R¹ R² delphinidin OH OH cyanidin OH H malvidin OCH₃ OCH₃ pelargonidin HH peonidin OCH₃ H petunidin OCH₃ OH

[0004] Examples of anthocyanidin, i.e., an aglycon, include delphinidin,cyanidin, malvidin, pelargonidin, peonidin, and petunidin. Anthocyaninis referred to as anthocyanidin glucoside when, for example, glucose isbound to the anthocyanidin as a glycoside. Saccharide found inanthocyanin includes: monosaccharide such as galactose and arabinose inaddition to glucose; and disaccharide such as rutinose and sophorose.

[0005] Anthocyans are widely present in nature, and are mainly used as anatural pigment for food or, because of their functionalities, areextensively used for pharmaceuticals, quasi drugs, cosmetics, and thelike in Europe. For example, use thereof as a cicatrizant, as disclosedin Japanese Examined Patent Publication (Kokoku) No. 59-53883, orpharmacological properties thereof which are valuable in the treatmentof peripheral blood diseases using anthocyanin derived from blueberry,as disclosed in Japanese Laid-open Patent Publication (Kokai) No.3-81220, have been discovered. In recent years, the functionality ofanthocyanin has drawn attention in Japan for uses of anthocyanin otherthan as a pigment. The present inventors have also found several usefulefficacies in anthocyanin of black currant and these are reported in WO01/01798.

[0006] When these anthocyanins having pharmacological properties areused as pharmaceuticals and the like, highly purified ones are required.Heretofore, however, mass production of highly purified anthocyanins hasnever been realized. Further, while highly purified anthocyanins arepreferably crystalline from the viewpoint of stability, hygroscopicity,and the like, mass production of crystalline anthocyanins has likewisenot been realized up to now.

[0007] Conventionally, anthocyanin compositions for pharmaceuticals aremainly preparations derived from blueberry with an anthocyanin contentof 25% by weight or lower. Thus, at least several hundred mg of ananthocyanin preparation had to be administered per dose in order toexhibit its effectiveness, and the consumption of a small amount thereofcould not produce pharmacological effects in practice. Accordingly,compositions containing highly purified anthocyanin at high levels havebeen awaited.

[0008] Highly purified anthocyanin was not present because of thefollowing reasons. For example, in the case of blueberry-derivedanthocyanin, there are 15 types of anthocyanin components, and thephysiochemical properties of these substances are very similar to oneanother. Thus, the respective flux peaks thereof overlap with oneanother in purification using a preparative column or the like. Or,separation and purification were impossible because each component wasin a very small amount.

[0009] In the case of anthocyanin derived from black currant, forexample, four components, i.e., cyanidin-3-O-glucoside (hereinafter itis abbreviated to “C3G”), cyanidin-3-O-rutinoside (hereinafter it isabbreviated to “C3R”), delphinidin-3-O-glucoside (hereinafter it isabbreviated to “D3G”), and delphinidin-3-O-rutinoside (hereinafter it isabbreviated to “D3R”), are contained as anthocyanins. As with theblueberry-derived anthocyanin, due to very similar physiochemicalproperties among the four substances, even mixtures of these fourcomponents have very close chromatography peaks. Even if preparativechromatography or centrifugal partition chromatography were performed toobtain purified anthocyanin, mass production thereof was impossible dueto extremely deteriorated yield. The quantitative ratio ofrepresentative anthocyanins in black currant is as follows: D3G, D3R,C3G, and C3R are respectively present at 12.5%, 47.9%, 4.1%, and 35.5%.Consequently, purification of a large amount of D3G and C3G components,which are contained at low levels, involved further difficultly, andthus a process for mass producing purified anthocyanin has been awaited.

[0010] In contrast, anthocyan has been heretofore known to have adrawback in its stability. The present inventors have applied for patenton a process for stabilizing substances containing anthocyanin at highlevel by adding phytic acid, saccharides, and sugar alcohols asstabilizers (PCT/JP00/09204). However, when a large amount ofanthocyanin is used for making preparations, there is no room to containthese additives. Accordingly, more stable physical properties wererequired and preparations using crystalline anthocyanin, which isphysically more stable, were awaited. Thus, conventionally, high purityanthocyanin was organically synthesized for pharmaceutical applicationsthrough many steps by, for example, a process for synthesizingdelphinidin hydrochloride (anthocyanidin hydrochloride of an aglyconinstead of glycoside) as disclosed in Japanese Patent No. 3030509,although mass production thereof from natural products was not realized.

[0011] The anthocyanidin hydrochloride produced by the synthesis methodis stable under strong acidic conditions, however, it is likely to bedegraded as compared to a glycoside in weak acidic to neutral regions.The application range was thus very narrow. Accordingly, production ofanthocyanin which was more stable in acidic to neutral regions ascrystals was awaited, although mass production of anthocyanin by theorganic synthesis process is currently still unavailable.

[0012] Features of anthocyanins are listed in the Dictionary of NaturalProducts (issued by Chapman & Hall, 1994, London). For example, thecrystal of D3R has not been heretofore reported, and while the crystalform of D3G hydrochloride has been reported, its melting point is notdescribed. This indicates that mass production thereof was difficult.Similarly, although the melting point and the crystal form of C3Rhydrochloride are described, there is no description on the meltingpoint of C3G hydrochloride. This indicates that mass production thereofwas also difficult. Up to the present, only a very small amount ofpurified anthocyanin could be produced, regardless of whether it iscrystalline or not. Thus, there was substantially no study whichinvestigated the reactivity of various enzymes to anthocyanin.Especially, there was no description on the reactivity of rhamnosidaseto anthocyanin. Accordingly, a process for mass producing highlypurified anthocyanin from natural products without using complicatedsynthesis processes has been awaited. Further, a process for massproducing crystalline anthocyanin salts by crystallizing purifiedanthocyanin was also awaited.

SUMMARY OF THE INVENTION

[0013] One aspect of the present invention relates to a process forproducing purified anthocyanin in which a rhamnose end of anthocyanidinrutinoside is cleaved using rhamnosidase to convert the anthocyanidinrutinoside component into anthocyanidin glucoside, the anthocyanidinglucoside component being then purified and isolated, or a process forproducing crystalline anthocyanidin glucoside salt hydrate by furthercrystallizing the purified anthocyanin.

[0014] Another aspect of the present invention relates to a process forproducing purified anthocyanin in which a glucose end of anthocyanidinglucoside is cleaved using β-glucosidase to degrade and remove the end,the anthocyanidin rutinoside component being then purified and isolated,or a process for producing crystalline anthocyanidin rutinoside salthydrate by further crystallizing the purified anthocyanin.

[0015] A further aspect of the present invention relates to acrystalline anthocyanin salt hydrate prepared by these productionprocesses.

[0016] More specifically, the first invention provides a process forproducing purified anthocyanidin glucoside, wherein rhamnosidase isallowed to act upon an anthocyanin composition containing at least onekind of anthocyanidin rutinoside, and anthocyanidin rutinoside issubjected to hydrolysis to convert into anthocyanidin glucoside, whichis then isolated and purified.

[0017] The second invention provide a process for producing purifiedanthocyanidin rutinoside, wherein β-glucosidase is allowed to act uponan anthocyanin composition containing at least one kind of anthocyanidinglucoside and anthocyanidin rutinoside, and anthocyanidin glucoside issubjected to hydrolysis to reduce the anthocyanidin glucoside, theanthocyanidin rutinoside being then isolated and purified.

[0018] In the aforementioned first and second inventions, theanthocyanin composition includes fruit juice obtained from at least onemember selected from black currant, fig, coffee, banana, blackberry, andthe like, and/or an anthocyanin concentrate obtained from wild rice(Zizania aquatica Linn.), colocasia, and the like. The rhamnosidaseincludes hesperidinase, naringinase and the like. Further, theβ-glucosidase can selectively degrade only the β-glucoside bond ofanthocyanidin glucoside without degrading the β-glucoside bond ofanthocyanidin rutinoside, and specific examples thereof includeβ-glucosidase derived from almond.

[0019] The third invention provides a process for producing crystallineanthocyanidin-3-O-glucoside hydrochloride hydrate through the steps of:

[0020] a) allowing rhamnosidase to act upon an anthocyanin compositioncontaining at least one kind of anthocyanidin rutinoside and subjectinganthocyanidin rutinoside to hydrolysis to convert into anthocyanidinglucoside;

[0021] b) purifying the anthocyanidin glucoside to obtain anthocyanidinglucoside of 99% or higher purity; and

[0022] c) crystallizing the anthocyanidin glucoside using a mixedsolvent of hydrochloric acid/alcohol system.

[0023] The fourth invention provides a crystallineanthocyanidin-3-O-glucoside hydrochloride hydrate obtained by the methodaccording to the third invention.

[0024] The fifth invention provides a process for producing crystallineanthocyanidin-3-0rutinoside hydrochloride hydrate through the steps of:

[0025] a) allowing β-glucosidase to act upon an anthocyanin compositioncontaining at least one kind of anthocyanidin glucoside andanthocyanidin rutinoside, and subjecting anthocyanidin glucoside tohydrolysis to reduce the anthocyanidin glucoside;

[0026] b) purifying the anthocyanidin rutinoside to obtain anthocyanidinrutinoside of 99% or higher purity; and

[0027] c) crystallizing the anthocyanidin rutinoside using a mixedsolvent of hydrochloric acid/alcohol system.

[0028] The sixth invention provides a crystallineanthocyanidin-3-O-rutinoside hydrochloride hydrate obtained by themethod according to the fifth invention.

[0029] In the third and the fifth inventions, the purification processaccording to step b) can be carried out by ion exchange adsorptionchromatography and/or HPLC, and the mixed solvent of hydrochloricacid/alcohol system used in step c) can be composed of 5% (v/v)hydrochloric acid/95% (v/v) methanol.

[0030] The seventh invention provides a crystallinedelphinidin-3-O-glucoside hydrochloride 0.5 hydrate having the followingphysical properties:

[0031] Melting point based on thermoanalysis: 258° C.

[0032] Uvλ max(ε): 517 nm (27500)

[0033] FAB-MS m/z:M⁺:465 Compositional formula: C₂₁H₂₁O₁₂Cl.0.5H₂OElementary analysis: C H Cl Measured values: 48.00 4.50 6.80

[0034] The eighth invention provides a crystallinecyanidin-3-O-glucoside hydrochloride 0.5 hydrate having the followingphysical properties:

[0035] Melting point based on thermoanalysis: 245° C.

[0036] Uvλ max(ε): 510 nm (26300)

[0037] FAB-MS m/z:M⁺:449 Compositional formula: C₂₁H₂₁O₁₁Cl.0.5H₂OElementary analysis: C H Cl Measured values: 48.80 4.70 6.90

[0038] The ninth invention provides a crystallinedelphinidin-3-O-rutinoside hydrochloride 1.5 hydrate having thefollowing physical properties:

[0039] Melting point based on thermoanalysis: 224° C.

[0040] Uvλ max(ε):520 nm (27800)

[0041] FAB-MS m/z M⁺:611 Compositional formula: C₂₇H₃₁O₁₆Cl.1.5H₂OElementary analysis: C H Cl Measured values: 45.80 5.30 5.20

[0042] The tenth invention provides a crystallinecyanidin-3-O-rutinoside hydrochloride 0.5 hydrate having the followingphysical properties:

[0043] Melting point based on thermoanalysis: 214 to 226° C.

[0044] Uvλ max(ε):512 nm (27400)

[0045] FAB-MS m/z: M⁺:595 Compositional formula: C₂₇H₃₁O₁₅Cl.0.5H₂OElementary analysis: C H Cl Measured values: 50.00 5.30 5.30

[0046] The term “anthocyanin” used herein refers to a glycoside whichwas prepared by the binding of saccharides to anthocyanidin, i.e., anaglycon, and examples thereof include glucoside, rutinoside,arabinoside, and galactoside to which saccharides such as glucose,rutinose, arabinose, and galactose have been bonded.

[0047] In the present invention, anthocyanin compositions containing atleast one kind of anthocyanidin glucoside and/or anthocyanidinrutinoside that are used in the enzyme reaction may be any substanceswhich contain anthocyanin. Examples thereof include commerciallyavailable fruit juice, concentrated juice, beverages, pigment solutions,and powders which are starting materials for foods, pharmaceuticals, orthe like. Preferably, concentrated juice is fruit juice obtained from atleast one member selected from black currant, fig, coffee, banana,blackberry, and the like, and/or an anthocyanin concentrate which isobtained from wild rice (Zizania aquatica Linn.), colocasia, and thelike. The powders are first dissolved in water, buffer, or the like touse the solution in the reaction. More preferably, compositionscontaining anthocyanin at high levels which are prepared by the methodreported in WO 01/01798 by the present inventors are used. Since acids,saccharides, polyphenols, and the like are previously removed, operationin the purification step after the enzyme reaction is facilitated.

[0048] The types of aglycon of anthocyanin used for the process forproducing purified anthocyanin according to the present invention arenot particularly limited. Black currant-derived anthocyanins and saltsthereof are preferred. An aglycon moiety of the black currant-derivedanthocyanin consists only of delphinidin and cyanidin. In the enzymereaction, however, these two types are not considered to be particularlyspecific for anthocyanidin, and can be applied to a glycoside of anotheranthocyanidin, for example, glycosides such as malvidin, pelargonidin,petunidin, and peonidin represented by formula (I). Further, the blackcurrant-derived anthocyanin component is constituted by four components:cyanidin-3-O-glucoside (C3G), cyanidin-3-O-rutinoside (C3R),delphinidin-3-O-glucoside (D3G), and delphinidin-3-O-rutinoside (D3R).

[0049] The types of salts which constitute crystalline anthocyanin saltinclude salts with mineral acids such as hydrochloric acid and sulfuricacid and salts with organic acids (flavinium salt) such as phosphoricacid, tritluoroacetic acid (TFA), and acetic acid. From the viewpoint ofeasy crystallization, salts with hydrochloric acid, TFA, and phosphoricacid are preferable.

[0050] Rhamnosidase that is used in the present invention is notparticularly limited, and it may originate from animals, plants,microorganisms, and the like as long as it has α-rhamnosidase activitiesat the anthocyanin end as the action mechanism. However, it is importantthat it has low β-glucosidase activities. Further, anthocyanin is knownto be degraded when it is allowed to stand in neutral to basic regionsfor a long period of time, and its degradation is accelerated byheating. Accordingly, it is necessary that the activity level is high atpH 4.0 or below and at 40° C. or below. Preferable examples thereofinclude hesperidinase or naringinase, with hesperidinase being morepreferred. Hesperidinase includes Aspergillus niger-derived substanceswhich are industrially produced for the purpose of degrading hesperidin,which is an opaque white component of Satsuma mandarins. The reactivityof Aspergillus niger-derived hesperidinase to anthocyanin has not beenheretofore known. Naringinase also includes Aspergillus niger-derivedsubstances which are used for taste improvement by degrading naringin,which is a bitter principle from abscised oranges or Citrus aurantium(natsu-mikan). It should be noted that the application of Aspergillusniger-derived naringinase to anthocyanin has not been reported.

[0051] The source of the β-glucosidase that is used in the presentinvention is not particularly limited. It must react with anthocyaninand it should have a substrate specificity to degrade only theβ-glucoside bond of anthocyanidin glucoside without degrading theβ-glucoside bond of anthocyanidin rutinoside. Specifically, suitableβ-glucosidase reacts with only terminal glucose but it does not reactwith glucose which is present at the center in the molecule. As withrhamnosidase, the activity level should be high at pH 4.0 or below andat 40° C. or below, and preferable examples thereof includealmond-derived β-glucosidase. While the almond-derived β-glucosidase isa representative β-glucosidase, there has been no study whichinvestigated the reactivity to anthocyanin.

[0052] Examples of treatments utilizing the reaction of β-glucosidase toanthocyanin include the conventional use of anthocyanase, which is atype of β-glucosidase, for decolorization and debittering in the fruitjuice industry for grape juice and peach nectar and the like, and in thewine industry for wines and sparkling wines including champagnes.However, anthocyanase reacts with and degrades both glucoside andrutinoside of anthocyanin. Thus, it is not suitable for the productionof purified anthocyanin.

[0053] The reaction conditions which allow these enzymes to act onanthocyanin are not particularly limited. As described above,anthocyanin is known to be degraded when it is allowed to stand inneutral to basic regions for a long period of time and its degradationis accelerated by high temperature. Thus, an acidic region ispreferable, and preferably, it is not reacted at high temperature.Specifically, reaction at pH 4.0 or below and at 40° C. or below islikely to be most preferable.

[0054] The substrate concentration in the anthocyanin-containingsubstance that is used in the enzyme reaction is not particularlylimited. When the concentration is extremely high, the viscosity of thereaction solution increases and the reaction speed is lowered. Due to afear of inhibitory reaction by substances in the reaction solution ortransition reaction to a substrate, the substrate concentration ispreferably 10% (w/v) or below, and more preferably 5% (w/v) or below.The amount of enzymes added is not particularly limited in relation tothe reaction time.

[0055] After the content of a substrate in the anthocyanin-containingsubstance, i.e., anthocyanidin rutinoside or anthocyanidin glucoside, isdecreased by the enzyme reaction using rhamnosidase or β-glucosidase,the enzyme reaction can be terminated by commonly employed methods, andexamples thereof include raising the temperature by heating, pHadjustment by adding acid or alkali, and addition of organic solvent. Asdescribed above, anthocyanins are unstable in neutral to basic regionsand their degradation is accelerated by heating. Thus, the enzymereaction is preferably terminated by pH lowering with the addition ofstrong acids such as hydrochloric acid, TFA, and phosphoric acid or bythe addition of organic solvent such as methanol.

[0056] In the present invention, purification after the completion ofthe enzyme reaction can be carried out by column chromatography as wellas by a suitable combination of another chromatography, resinadsorption, membrane separation, and the like, if necessary. Of all themethods, chromatography using ODS-silica gel is most preferable. Ifnecessary, the anthocyanin component may be first adsorbed on a cationexchange resin or the like and then eluted in order to similarly purifythe prepurified one.

[0057] The anthocyanin content herein is determined by summing thecontents of each of the anthocyanin components contained, which arecalculated by the peak area ratio of anthocyanin by HPLC, as with thedisclosure in PCT/JP 00/09204 and WO 01/01798.

[0058] Specifically, an anthocyanin sample with a known weight is firstanalyzed by HPLC and the calibration curve is prepared based on the peakarea at 520 nm, to thereby determine the content of each anthocyanincomponent. Further, the content of each of the components is divided bythe peak area to determine the response coefficient, i.e., the mg/peakarea. Subsequently, the anthocyanin-containing sample is subjected toHPLC analysis, the response coefficient which was determined from thesample is multiplied by the peak area of each component to calculate thecontent of each component. Thus, the anthocyanin purity is determined as% by weight (w/w) based on the ratio with the amount injected.Accordingly, the purity of anthocyanin is calculated by including theamount of a bound saccharide in addition to the amount of anthocyanidin,i.e., an aglycon. The purity level of the purified anthocyanin accordingto the present invention is 99% or higher based on the HPLC analysis.

[0059] The crystallization process according to the present invention ispreferably carried out mainly from an organic solvent, and methanol ispreferably used as the crystallizing solvent. Because anthocyanins arecrystallized by generating salts with acids, the addition of acids inthe crystallizing solvent is preferred. Preferably, about 1% to 5% (v/v)of hydrochloric acid, TFA, phosphoric acid, or the like is added.

[0060] The crystalline D3C, crystalline D3R, crystalline C3G, andcrystalline C3R which were prepared in Example 1 or 3 below weresubjected to thermoanalysis, and the results thereof are provided below.

[0061] The melting point of D3G is 258° C., that of D3R is 224° C., thatof C3G is 245° C., and that of C3R is 214 to 226° C.

[0062] The crystalline anthocyanin salt according to the presentinvention is crystal having 99% or higher purity based on thepolarization microscopy, elementary analysis, melting pointdetermination, and HPLC analysis, and is a very stable substance withouthygroscopicity and with a melting point of 200° C. or higher.

[0063] In the past, highly purified anthocyanin or crystallineanthocyanin salt could not be produced. With the use of the productionprocess according to the present invention, however, highly purifiedanthocyanin and crystalline anthocyanin salt could be produced throughpurification from natural products. The thus produced crystallineanthocyanin salts did not exhibit any hygroscopicity and were stable.

BEST MODE FOR CARRYING OUT THE INVENTION

[0064] The present invention will be described in more detail withreference to the following examples, reference examples, and comparativeexamples. The technical scope of the present invention, however, is notlimited by these examples.

REFERENCE EXAMPLE 1

[0065] Preparation of Composition Containing Anthocyanin at High Level

[0066] In accordance with the process as described in WO 01/01798,compositions containing anthocyanin at high level were prepared.Specifically, 3 kg of commercially available concentrated black currantjuice (the anthocyanin purity per solid content: 2.8%) was diluted withwater to prepare diluted fruit juice with a concentration of Bx 10. Thediluted fruit juice was filtered with a filter paper to remove foreignmatter. Thereafter, membrane separation was carried out using a membraneseparator (NTR-7410, Nitto Denko Co., Ltd.). The concentrated liquidobtained by membrane separation was spray dried to obtain a powderycomposition containing anthocyanin at high level. The anthocyanin purityof this composition was 14.1% per solid content. This compositionexhibited hygroscopicity when it was allowed to stand at roomtemperature.

EXAMPLE 1

[0067] Production of Purified Delphinidin-3-O-glucoside and PurifiedCyanidin-3-O-glucoside Using Hesperidinase

[0068] The powder obtained in Reference Example 1 (40 g) (anthocyaninpurity: 14.1%; proportion of each anthocyanin component: 12.5%, 47.9%,4.1%, and 35.5% for D3G, D3R, C3G, and C3R, respectively) was dissolvedin 1 liter of 50 mM acetate buffer (pH 3.5) to prepare an anthocyaninsubstrate solution. The calculated anthocyanin content in the substratesolution was 5.64 g, and the contents of D3G, D3R, C3G, and C3R were0.71 g, 2.70 g, 0.23 g, and 2.00 g, respectively.

[0069] Separately, 79.35 g of hesperidinase (tradename: HesperidinaseTanabe 2, Tanabe Seiyaku Co,. Ltd) was dissolved in 1 liter of 50 mMacetate buffer (pH 3.5) to prepare an enzyme solution (corresponding tothe rhamnosidase activity of 42.5 U/ml).

[0070] The substrate solution and the enzyme solution were respectivelyheated at 40° C. and were then mixed to initiate the reaction. Thereaction was carried out at 40° C. for 6 hours, and 2 liters of 3% (w/v)phosphoric acid solution was added to terminate the reaction.Subsequently, 4 liters of ion exchange resin XAD-7 (Rohm and HaasCompany) was filled into a column (13 cm (inside diameter)×30 cm(length)), and the reaction mixture (4 liters) was passed therethroughto allow the anthocyanin component to adsorb thereon. Subsequently, 0.1%(w/v) TFA (2 liters) was allowed to pass therethrough to wash thenonadsorbed component. Further, an 80% (v/v) aqueous methanol solutioncontaining 0.1% TFA was passed therethrough to eluate the adsorbedcomponent. This methanol solution was concentrated using a rotaryevaporator and converted into an aqueous 3% phosphoric acid solution tobring the concentration of the solid content to 10% (w/v). Thus, aconcentrated liquid was obtained.

[0071] This concentrated liquid was detected using an ODS-120T silicagel column (ED 5.5×30 cm, 20 μm, TOSOH CORPORATION) with an aqueous 9%(v/v) acetonitrile solution containing 0.1% TFA at a flow rate of 80ml/min at a wavelength of 520 nm to obtain the D3G fraction with aretention time (R.T.) of 66 to 90 min and the C3G fraction with an R.T.of 158 to 200 min. These fractions has a single peak based on the HPLCanalysis, and purified delphinidin-3-O-glucoside and purifiedcyanidin-3-O-glucoside with a purity level of 99% or higher could beobtained.

[0072] Conditions for the HPLC analysis to measure the purity are asfollows. Specifically, the analysis was performed under the followinggradient conditions using the Hewlett Packard Series 1100 HPLC System(Yokogawa Analytical Systems Inc.).

[0073] HPLC gradient conditions: Liquid A (aqueous 0.5% Time (min)phosphoric acid solution) Liquid B (methanol) 0 80 20 15 77 23 20 77 2330 50 50 40 50 50

[0074] A Zorbax SB-C18 column (4.6 mm×250 nm, 5 μm, Hewlett Packard) wasused. Detection was carried out at the flow rate of 1 ml/min at thewavelength of 520 nm. The R.T.s of the sample D3G and C3G wererespectively 10.54 min and 14.60 min.

[0075] A part of the substrate solution before the enzyme reaction and apart of the solution in which the enzyme reaction was terminated werecollected, and foreign matters were removed through a microfilter with apore diameter of 0.45 μm to prepare an anthocyanidin glucoside solution.The contents of anthocyanin components before the reaction and after thereaction were measured. The results are as shown in Table 1. The resultsindicate that the substrate solution consisted only of anthocyanidinglucoside because anthocyanidin rutinoside was degraded andanthocyanidin glucoside was generated in the substrate solution. Inaddition, the amount of D3G was 3.36 times higher than before thereaction, and the amount of C3G was 6.78 times higher than before thereaction. TABLE 1 Change in anthocyanin content D3G D3R C3G C3R Beforereaction 0.71 g 2.70 g 0.23 g 2.00 g After reaction 2.39 g 0.00 g 1.56 g0.00 g

EXAMPLE 2

[0076] Production of Crystalline Delphinidin-3-O-glucoside HydrochlorideHydrate and Crystalline Cyanidin-3-O-glucoside Hydrochloride Hydrate

[0077] The D3G fraction and C3G fraction obtained in Example 1 wereconcentrated using a rotary evaporator, and 30 ml of heptane was addedthereto, followed by reconcentration to dryness. TFA which was includedduring the separation operation was removed. The result of weightmeasurement showed the obtained D3G fraction was 1.51 g and the C3Gfraction was 0.98 g.

[0078] The D3G fraction and the C3G fraction were separately dissolvedin 5% hydrochloric acid/95% methanol, and were then allowed to stand at5° C. for 24 hours to perform crystallization. Solid liquid separationwas carried out using the Kiriyama funnel and No. 2 filter paper(Whatman), and washing with a small amount of acetone was carried out,followed by drying to obtain precipitates. Both of the obtainedprecipitates were observed under a polarization microscope, andpolarization of light was observed. This indicated that they were incrystal forms. The yield of crystalline D3G hydrochloride was 1.06 g andthat of crystalline C3G hydrochloride was 0.59 g.

[0079] The structures of the crystalline D3G hydrochloride and thecrystalline C3G hydrochloride obtained were determined by NMR. Thestructures of these two types of anthocyanins were consistent with thespectrum data which have been already reported.

[0080] Other physical properties of the crystalline D3G hydrochlorideand the crystalline C3G hydrochloride are as follows.

[0081] Crystalline D3G hydrochloride:

[0082] Melting point based on thermoanalysis: 258° C.

[0083] Uvλ max(ε): 517 nm (27500)

[0084] FAB-MS m/z: M⁺:465 Compositional formula: C₂₁H₂₁O₁₂Cl.0.5H₂OElementary analysis: C H Cl Measured values: 48.00 4.50 6.80 Calculatedvalues 47.78 4.58 6.72

[0085] Crystalline C3G hydrochloride:

[0086] Melting point based on thermoanalysis: 245° C.

[0087] Uvλ max(ε): 510 nm (26300)

[0088] FAB-MS m/z: M⁺:449 Compositional formula: C₂₁H₂₁O₁₁Cl.0.5H₂OElementary analysis: C H Cl Measured values: 48.80 4.70 6.90 Calculatedvalues: 48.57 4.73 6.93

EXAMPLE 3

[0089] Production of Purified Delphinidin-3-O-rutinoside and PurifiedCyanidin-3-O-rutinoside Using Almond-Derived β-glucosidase

[0090] The powder (3.42 g) obtained in Reference Example 1 was dissolvedin 1 liter of 50 mM acetate buffer (pH 3.5) to prepare an anthocyaninsubstrate solution.

[0091] Separately, 208 g of almond-derived 0-glucosidase (SIGMA) wasdissolved in 1 liter of 50 mM acetate buffer (pH 3.5) to prepare anenzyme solution (corresponding to 500 U/ml). The substrate solution (1liter) was heated at 40° C. for 10 minutes to stabilize the temperature.Thereafter, 1 liter of enzyme solution was added and thoroughly stirredto initiate the reaction. Sixty minutes later, 2 liters of 0.3Nhydrochloric acid was added to terminate the reaction.

[0092] Subsequently, 4 liters of reaction mixture, in which the reactionwas terminated, was treated by the adsorption of an ion exchange resinin the same manner as described in Example 1, and further purified byHPLC using an ODS-silica gel column. Thus, a D3R fraction with an R.T.of 69 to 96 minutes and a C3R fraction with an R.T. of 144 to 174minutes were obtained.

[0093] These fractions were analyzed under the HPLC analysis conditionsas described in Example 1 (the R.T.s of the sample D3R and C3R were12.63 minutes and 18.19 minutes, respectively). The results showed thatthe D3R fraction and the C3R fraction had a single peak. Thus, purifieddelphinidin-3-O-rutinoside and purified cyanidin-3-O-rutinoside having apurity level of 99% or higher could be obtained.

[0094] The anthocyanin contents in the substrate solution before thereaction and that in the solution after the reaction which were measuredby HPLC are shown in Table 2. According to Table 2, only anthocyanidinglucoside was degraded and most of anthocyanidin rutinoside remainedundegraded. This indicates that a reaction solution having an optimalcomposition for purification was obtained. TABLE 2 Change in anthocyanincontent D3G D3R C3G C3R Before reaction 60.3 mg 231 mg 19.8 mg 171 mgAfter reaction  5.6 mg 207 mg  0.0 mg 162 mg

EXAMPLE 4

[0095] Production of Crystalline Delphinidin-3-O-rutinosideHydrochloride Hydrate and Crystalline Cyanidin-3-O-rutinosideHydrochloride Hydrate

[0096] Precipitates were obtained through the crystallization process aswith Example 2. Both the obtained precipitates were observed under apolarization microscope, and as a result, polarization of light wasobserved and they were found to be in crystal forms. The yield ofcrystalline C3R hydrochloride was 58 mg and that of crystalline D3Rhydrochloride was 88 mg.

[0097] The structures of the crystalline D3R hydrochloride and thecrystalline C3R hydrochloride obtained were determined by NMR. Thestructures of these two types of anthocyanins were consistent with thespectrum data which have been already reported.

[0098] Physical properties of the crystalline D3R hydrochloride and thecrystalline C3R hydrochloride are as follows.

[0099] Crystalline D3R hydrochloride:

[0100] Melting point based on thermoanalysis: 224° C.

[0101] Uvλ max(ε): 520 nm (27800)

[0102] FAB-MS m/z: M⁺:611 Compositional formula: C₂₇H₃₁O₁₆Cl.1.5H₂OElementary analysis: C H Cl Measured values: 45.80 5.30 5.20 Calculatedvalue: 45.86 5.38 4.98

[0103] Crystalline C3R hydrochloride:

[0104] Melting point based on thermoanalysis: 214 to 226° C.

[0105] Uvλ max(ε): 512 nm (27400)

[0106] FAB-MS m/z: M⁺:595 Compositional formula: C₂₇H₃₁O₁₅Cl.0.5H₂OElementary analysis: C H Cl Measured values: 50.00 5.30 5.30 Calculatedvalues: 49.97 5.13 5.46

COMPARATIVE EXAMPLE 1

[0107] Enzyme Reaction Using Commercially Available Anthocyanase

[0108] The powder (342 mg) obtained in Reference Example 1 was dissolvedin 100 ml of 50 mM acetate buffer (pH 3.5) to prepare an anthocyaninsubstrate solution.

[0109] Separately, CYTOLASE PCL5 (tradename, GIST-brocades), which is atype of representative anthocyanase used in the fruit juice industry,was diluted to 10-fold with a 50 mM acetate buffer (pH 3.5) to obtain anenzyme solution.

[0110] The substrate solution (2 ml) was heated at 40° C. for 10 minutesto stabilize the temperature. Thereafter, 2 ml of enzyme solution wasadded and thoroughly stirred to initiate the reaction. Fifteen minuteslater, 200 μl of a reaction solution was sampled, and 200 μl of 0.3Nhydrochloric acid was added to terminate the reaction.

[0111] The anthocyanin compositions before the reaction and after thereaction were measured by HPLC. The anthocyanin contents andcompositions before and after the reaction are shown in Table 3 below.Table 3 shows that both the anthocyanidin glucoside and theanthocyanidin rutinoside were degraded, indicating the composition wasunsuitable for purification of anthocyanin glycoside. TABLE 3 Change inanthocyanin content D3G D3R C3G C3R Before reaction 0.60 mg 2.31 mg 0.20mg 1.71 mg After reaction 0.23 mg 0.09 mg 0.26 mg 0.04 mg

[0112] Industrial Applicability

[0113] The present invention enabled the production of highly purifiedanthocyanin and crystalline anthocyanin salt through purification fromnatural products. The thus produced crystalline anthocyanin salt did notexhibit hygroscopicity and was stable.

[0114] This specification includes part or all of the contents asdisclosed in the specification of Japanese Patent Application No.2000-276540, which is a priority document of the present application.All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

1. A process for producing purified anthocyanidin glucoside, whereinrhamnosidase is allowed to act upon an anthocyanin compositioncontaining at least one kind of anthocyanidin rutinoside to convertanthocyanidin rutinoside into anthocyanidin glucoside, which is thenisolated and purified.
 2. The process for producing purifiedanthocyanidin glucoside according to claim 1, wherein the anthocyanincomposition is fruit juice obtained from at least one member selectedfrom black currant, fig, coffee, banana, and blackberry and/or ananthocyanin concentrate obtained from wild rice (Zizania aquatica Linn.)or colocasia.
 3. The process for producing purified anthocyanidinglucoside according to claim 1 or 2, wherein the rhamnosidase ishesperidinase or naringinase.
 4. A process for producing purifiedanthocyanidin rutinoside, wherein β-glucosidase is allowed to act uponan anthocyanin composition containing at least one kind of anthocyanidinglucoside and anthocyanidin rutinoside to reduce anthocyanidinglucoside, the anthocyanidin rutinoside being then isolated andpurified.
 5. The process for producing purified anthocyanidin rutinosideaccording to claim 4, wherein the anthocyanin composition is fruit juiceobtained from at least one member selected from black currant, fig,coffee, banana, and blackberry and/or an anthocyanin concentrateobtained from wild rice (Zizania aquatica Linn.) or colocasia.
 6. Theprocess for producing purified anthocyanidin rutinoside according toclaim 4 or 5, wherein the β-glucosidase can selectively degrade only theβ-glucoside bond of anthocyanidin glucoside without degrading theβ-glucoside bond of anthocyanidin rutinoside.
 7. The process forproducing purified anthocyanidin rutinoside according to any one ofclaims 4 to 6, wherein the β-glucosidase is derived from almond.
 8. Aprocess for producing crystalline anthocyanidin-3-O-glucosidehydrochloride hydrate through the steps of: a) allowing rhamnosidase toact upon an anthocyanin composition containing at least one kind ofanthocyanidin rutinoside to convert anthocyanidin rutinoside intoanthocyanidin glucoside; b) purifying the anthocyanidin glucoside toobtain anthocyanidin glucoside of 99% or higher purity; and c)crystallizing the anthocyanidin glucoside using a mixed solvent ofhydrochloric acid/alcohol system.
 9. The process for producingcrystalline anthocyanidin-3-O-glucoside hydrochloride hydrate accordingto claim 8, wherein the purification process according to step b) inclaim 8 is carried out by ion exchange adsorption chromatography and/orHPLC.
 10. The process for producing crystallineanthocyanidin-3-O-glucoside hydrochloride hydrate according to claim 8or 9, wherein the mixed solvent of hydrochloric acid/alcohol system iscomposed of 5% (v/v) hydrochloric acid/95% (v/v) methanol. 11.Crystalline anthocyanidin-3-O-glucoside hydrochloride hydrate, which isobtained by the process according to any one of claims 8 to
 10. 12. Aprocess for producing crystalline anthocyanidin-3-O-rutinosidehydrochloride hydrate through the steps of: a) allowing β-glucosidase toact upon an anthocyanin composition containing at least one kind ofanthocyanidin glucoside and anthocyanidin rutinoside to reduceanthocyanidin glucoside; b) purifying the anthocyanidin rutinoside toobtain anthocyanidin rutinoside of 99% or higher purity; and c)crystallizing the anthocyanidin rutinoside using a mixed solvent ofhydrochloric acid/alcohol system.
 13. The process for producingcrystalline anthocyanidin-3-O-rutinoside hydrochloride hydrate accordingto claim 12, wherein the purification process according to step b) inclaim 12 is carried out by ion exchange adsorption chromatography and/orHPLC.
 14. The process for producing crystallineanthocyanidin-3-O-rutinoside hydrochloride hydrate according to claim 12or 13, wherein the mixed solvent of hydrochloric acid/alcohol system iscomposed of 5% (v/v) hydrochloric acid/95% (v/v) methanol. 15.Crystalline anthocyanidin-3-O-rutinoside hydrochloride hydrate, which isobtained by the process according to any one of claims 12 to
 14. 16.Crystalline delphinidin-3-O-glucoside hydrochloride 0.5 hydrate havingthe following physical properties: Melting point based onthermoanalysis: 258° C. Uvλ max(ε): 517 nm (27500) FAB-MS m/z: M⁺:465Compositional formula: C₂₁H₂₁O₁₂Cl.0.5H₂O Elementary analysis: C H ClMeasured values: 48.00 4.50 6.80


17. Crystalline cyanidin-3-O-glucoside hydrochloride 0.5 hydrate havingthe following physical properties: Melting point based onthermoanalysis: 245° C. Uvλ max(ε): 510 nm (26300) FAB-MS m/z: M⁺:449Compositional formula: C₂₁H₂₁O₁₁Cl.0.5H₂O Elementary analysis: C H ClMeasured values: 48.80 4.70 6.90


18. Crystalline delphinidin-3-O-rutinoside hydrochloride 1.5 hydratehaving the following physical properties: Melting point based onthermoanalysis: 224° C. Uvλ max(ε): 520 nm (27800) FAB-MS m/z: M⁺:611Compositional formula: C₂₇H₃₁O₁₆Cl.1.5H₂O Elementary analysis: C H ClMeasured values: 45.80 5.30 5.20


19. Crystalline cyanidin-3-O-rutinoside hydrochloride 0.5 hydrate havingthe following physical properties: Melting point based onthermoanalysis: 214 to 226° C. Uvλ max(ε): 512 nm (27400) FAB-MS m/z:M⁺:595 Compositional formula: C₂₇H₃₁O₁₅Cl.0.5H₂O Elementary analysis: CH Cl Measured values: 50.00 5.30 5.30